1. Field of the Invention
The present invention relates to an enzyme electrode and a method of determining concentration of an analyte in a sample solution by means of the enzyme electrode.
2. Description of the Prior Art
Many reports have been made up to the present day on an enzyme electrode comprising an electrochemical transducer device for detecting variation of phase boundary potential and an enzyme-immobilized membrane provided on the surface of the electrochemical transducer device.
For example, as to the enzyme electrode wherein glucose oxidasc is immobilized as the enzyme, that is, a glucose sensor, there are many embodiments already reported, such as those described in "Biophysica Biochimica Acta", Vol. 320 (1973), pages 529-534 and "Analytical Chemistry", Vol. 57 (1985), pages 1917-1925. Each of these sensors relies upon a principle that glucose produces gluconic acid by catalytic action of glucose oxidase and the thus produced gluconic acid can be determined by measuring pH variation. The reaction procedures to produce gluconic acid from glucose may be shown as follows: EQU glucose+O.sub.2 .fwdarw..delta.-gluconic lactone+H.sub.2 O.sub.2( 1) EQU .delta.-gluconic lactone+H.sub.2 O.fwdarw.gluconic acid (2)
The reaction (1) is catalyzed by glucose oxidase and the reaction (2) proceeds spontaneously or by catalytic action of gluconolactonase.
In the above mentioned glucose sensors, oxygen is required for operating them. Ordinarily, oxygen dissolved in the solution to be determined is used, but its concentration is to an extent of 0.25 mM in the case of saturation by atmosphere and of 1.2 mM in the case of saturation by oxygen gas and so is not sufficient for determining high concentration of glucose. Therefore, in order to determine high concentration of glucose, heretofore, a sample solution was diluted by a buffer solution and then determination was made. It was proposed, in order to make determination without such dilution, to use a glucose sensor which comprises an enzyme electrode, an enzyme-immobilized membrane and a permeation-restricted membrane, which has restricted permeability to glucose but has good permeability to oxygen, provided on the enzyme electrode, in Japanese Patent Appln. No. 5,171/1987. It was considered that by the merit of such permeation-restricted membrane, concentration of oxygen in the enzyme-immobilized membrane became high relatively to glucose concentration, and thus it would be possible to determine high concentration of glucose.
On the other hand, it is required in a glucose sensor utilizing an electrochemical transducer device detecting variation of phase boundary potential, particularly variation of hydrogen-ion concentration of a solution, that the solution to be analyzed has a constant or a known pH buffer capacity on a surface of the transducer device, in order to determine glucose concentration. The above permeation-restricted membrane however does not readily permeate molecules of substances which have pH buffer capacity in the solution to be analyzed, and so the enzyme-immobilized membrane is allowed to have only low pH buffer capacity and thus pH value in the enzyme-immobilized membrane is easily changed.
Therefore, in a glucose sensor utilizing an electrochemical transducer device detecting variation of phase boundary potential, the combination only of the enzyme-immobilized membrane and the permeation-restricted membrane is, in fact, not sufficient for determining high concentration of glucose, owing to a phenomenon that pH variation in the enzyme-immobilized membrane becomes larger and so an output of the sensor is saturated even by not so high concentration of glucose. Similar problem occurs in other sensors than the above glucose sensor as long as they require oxygen, such as an alcohol sensor utilizing a similar transducer device in combination with alcohol oxidase.
Further, in a method of determining concentration of an analyte in a sample solution by means of an enzyme electrode, there are problems of a complicated operation due to the fact that background output has to be corrected by immersing the electrode into a buffer solution which is free from glucose and also of a low S/N ratio of obtained signals due to the fact that adsorption of proteins or sudden change of pH value occurs when the electrode is immersed in a solution to be analyzed after the above correction procedure.